Hyperploidy (cells with abnormally high numbers of chromosomes) is observed in most human cancers and has been recognized as a hallmark of cancer cells for over a century. The association between hyperploidy and cancer is clear, and provides a clear therapeutic opportunity. If we can identify cellular processes strained or altered by hyperploidy, we can in principle develop therapies that specifically target hyperploid tumors. Currently, standard cancer chemotherapy agents such as taxol (paclitaxel) indiscriminately target dividing cells, causing myriad side effects that limit their efficacy and reduce patient quality of life. New cancer chemotherapies are needed, with greater selectivity for cancer cells. To perform its function, the mitotic spindle of a hyperploid cancer cell must align and segregate as many as four times the normal number of chromosomes. Hyperploid spindles are crowded with extra chromosomes and extra k-fibers (the bundles of microtubules that hold on to chromosomes), resulting in altered spindle architecture. Interestingly, not all k-fibers in hyperploid human cancer cells are directly connected to the spindle pole (Sikirzhytski* and Magidson* et al., 2014). Our laboratory recently discovered that chromosomes that lack a direct connection to the spindle pole can still undergo segregation via an alternative - now indirect - transport mechanism, mediated by the dynein-dynactin motor complex and the dynein adaptor protein NuMA (Elting* and Hueschen* et al., 2014). Here, we propose to investigate this newly- uncovered alternative mechanism of chromosome segregation and the role it plays in hyperploid cancer cells. We hypothesize that hyperploid cancer cells are preferentially dependent on this indirect chromosome segregation mechanism, and propose to investigate its underlying cellular biology with the goal of uncovering novel therapeutic targets and strategies. Specifically, we aim to identify the molecular mechanism of indirect chromosomes segregation and to determine if hyperploid cancer cells are preferentially dependent on indirect chromosome segregation for survival. Our strategy combines: spindle laser ablation as a tool to create indirect chromosome-to-pole connections, molecular perturbations and readouts, and high resolution microscopy of both non-transformed diploid cells and breast cancer cells of different ploidy. We expect that our investigation of the molecular mechanism of indirect chromosome segregation will reveal novel targets for cancer therapy. Moreover, we expect that uncovering the role of indirect segregation in hyperploid cell function will determine the potential for selective hyperploid cell death by indirect segregation inhibition, alone or in combination with existing cancer chemotherapies. In the long term, this work may contribute to the development of novel and selective cancer chemotherapies.